The protein tyrosine kinases Lyn and Syk play important roles in high affinity IgE receptor (FceRI)-induced signal transduction. Experiments with Syk negative cells indicate that most of the receptor-induced tyrosine phosphorylations are downstream of Syk and some of which may be actual substrates of this kinase. To identify novel substrates of Lyn and Syk, an RBL-2H3 bacterial expression library was screened for proteins that were tyrosine phosphorylated with baculoviral expressed Lyn and Syk. Five clones as potential Lyn substrates and eight clones as Syk substrates were identified including known substrates such as SLP-76, LAT, and alpha-tubulin. A potential substrate of Lyn identified was the molecule TOM1L1 which has several domains thought to be important for membrane trafficking and protein-protein interactions. Since the function of TOM1L1 is unclear, the rat TOM1L1 full-length cDNA was isolated and used to express the protein in COS-1 and RBL-2H3 cells. In COS-1 cells, the co-transfection of TOM1L1 and Lyn, but not Syk, resulted in the tyrosine phosphorylation of TOM1L1. In RBL-2H3 mast cells, both the endogenous and the over-expressed TOM1L1 were tyrosine phosphorylated in non-stimulated cells and this phosphorylation was increased by IgE receptor aggregation. By subcellular fractionation, wild-type TOM1L1 was mainly in the cytoplasm with a small fraction constitutively associated with the membrane; this association was markedly reduced in deletion mutants lacking several of the protein interaction domains. The over-expression of wild-type TOM1L1, but not the deletion mutants, enhanced the IgE-induced generation and release of cytokines. These results suggest that TOM1L1 is a novel protein involved in the FceRI signal transduction for the generation of cytokines. [unreadable] Immune receptor stimulation results in an increase in intracellular calcium that activates the serine phosphatase calcineurin, which then dephosphorylates the nuclear factor of activated T cells (NFAT). The dephosphorylated NFAT rapidly translocates into the nucleus and induces the transcription of various cytokine genes. NFAT plays an important role in the activation of immune cells such as T, B and mast cells. Therefore, NFAT can be used as readout for immune cell activation. A plasmid containing three tandem NFATbinding sites fused to the cDNA of enhanced green fluorescent protein (GFP) was transfected into the RBL-2H3 cells. The cells were then cloned and recloned to obtain a cell line that became almost totally GFP+ upon high affinity IgE receptor stimulation. Surprisingly with this sensitive detection system, there is activation of IgE sensitized cells at concentrations of antigen as low as 10 pg/ml, which is at least a log lower than can be detected by conventional methods. At this low antigen concentration there is no detectable release of inflammatory mediators which are the hallmarks of mast cell activation. However, NFAT activation at low antigen concentrations appears to utilize the same signaling pathways; for example the requirement for calcineurin. These results suggest differences between the pathways that result in nuclear gene expression from those that induce degranulation and the release of inflammatory mediators. These NFAT-GFP expressing cells are also being used to screen for regulators of the signaling pathway by transiently transfecting them with an RBL-2H3 cDNA plasmid library and testing for either inhibition or enhancement of the receptor-induced GFP response.[unreadable] Protein tyrosine phosphorylation is one of the earliest detectable events after antigen receptor activation. Recently, we observed that in cultured fetal liver pre-B cells, pre-B cell receptor stimulation induced the strong tyrosine phosphorylation of an 82 kDa protein, whose identity is unclear. The strong phosphorylation of this protein was also observed in antigen-receptor stimulated spleen B cells and mast cells. In mast cells, the phosphorylation of this 82 kDa protein required Syk kinase activity; however, this phosphorylation was independent of the rise of intracellular calcium. Therefore, the tyrosine phosphorylation of this 82 kDa protein directly correlated with early antigen signaling events in mast cells. To identify the 82 kDa phosphorylated protein, lysates from antigen stimulated RBL-2H3 cells were immunoprecipitated with anti-phospho-tyrosine antibody and the 82 kDa phosphorylated band was micro-sequenced. As a control, the phosphorylated 72 kDa band, that is known to contain Syk, was also micro-sequenced. The major component of the 82 kDa band was the hematopoietic cell specific Lyn substrate (HS1), while minor components included cortactin isoform B, inositol polyphosphate 5 phosphatase (SHIP), FYN binding protein, Gab 1, and protein kinase C-delta. The 72 kDa band, contained src homology 2 containing protein of 76 kDa (SLP-76), HS1, Syk, heat shock 70 protein, Btk and SHIP. There were a number of other proteins detected at lower levels in both the 82 and 70 kDa bands. Immunoblotting and immunoprecipitation experiments with control and activated RBL-2H3 cells confirmed that HS1 was the 82 kDa phospho-protein. Therefore, tyrosine phosphorylation of HS1 can serve as a marker for the functional status of early antigen signaling pathways.